Differential regulation of Ca²⁺ influx by fMLP and PAF in human neutrophils: Possible involvement of store-operated Ca²⁺ channel

Calcium (Ca²⁺) influx into human polymorphonuclear cells (PMNs) in response to N-formyl-Met-Leu-Phe (fMLP) and platelet-activating factor (PAF) stimulation was studied. Whole blood was taken by venous puncture from healthy human volunteers. PMNs were isolated, diluted, and incubated with 2 μM fura-2 AM. The cytosolic free calcium concentration, [Ca²⁺]_i, in human neutrophils was determined by microfluorometry. We found that the net area under the fMLP-or PAF-induced [Ca²⁺]_i rise curve in Ca²⁺-free medium decreased to 75% or 30% of the area under the curve in Ca²⁺ medium. Treatment of PMNs with phorbol myristate acetate (PMA), a protein kinase C activator, completely abolished the intracellular Ca²⁺ level stimulated by PAF, but not the intracellular Ca²⁺ level stimulated by fMLP. Treatment of PMNs with PAF did not abolish the intracellular Ca²⁺ level elevation stimulated by fMLP. In addition, treatment of PMNs with fMLP did not abolish intracellular Ca²⁺ level elevation stimulated by PAF. Loperamide, a positive modulator for store-operated calcium (SOC) channels, elicited an increase in intracellular calcium after the activation of SOC channels stimulated by fMLP or PAF. After the addition of guanosine 3′,5′-cyclic monophosphate, N²,2′-O-Dibutyryl-, sodium salt (db-cGMP), the initial increase of PAF-or fMLP-induced PMNs intracellular Ca²⁺ fluorescences was well preserved, but the slope and the peak height of fluorescence curves declined compared with the curves without db-cGMP. In conclusion, we found that PAF and fMLP regulate the Ca²⁺ influx of PMNs in different ways. Most of the PAF-induced [Ca²⁺]_i rise resulted from Ca²⁺ influx, and most of the fMLP-induced [Ca²⁺]_i elevation resulted from intracellular stores release. The initial mobilization of intracellular Ca²⁺ stores in PAF-stimulated signals is mediated by protein kinase C (PKC) phosphorylation, but not in fMLP-stimulated route. SOC channels are present and important in the fMLP-or PAF-induced PMNs Ca²⁺ influx. There was no apparent cross-regulation between PAF-and fMLP-stimulated intracellular Ca²⁺ influx.